WO2024055758A1 - Composé métallique dinucléotidique cyclique, son procédé de préparation et son utilisation - Google Patents

Composé métallique dinucléotidique cyclique, son procédé de préparation et son utilisation Download PDF

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WO2024055758A1
WO2024055758A1 PCT/CN2023/109735 CN2023109735W WO2024055758A1 WO 2024055758 A1 WO2024055758 A1 WO 2024055758A1 CN 2023109735 W CN2023109735 W CN 2023109735W WO 2024055758 A1 WO2024055758 A1 WO 2024055758A1
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cyclic dinucleotide
metal compound
group
cyclic
dinucleotide
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张跃茹
谭瀛轩
谭相宝
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杭州星鳌生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/213—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids containing cyclic phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

Definitions

  • the invention relates to a cyclic dinucleotide metal compound and its preparation method and application, and belongs to the field of biomedicine technology.
  • microbial and viral DNA can induce potent endogenous immune responses by stimulating interferon secretion.
  • Endoplasmic reticulum (ER) receptor protein (STING) is an essential factor in the immune response to cytosolic DNA.
  • ER Endoplasmic reticulum
  • STING Endoplasmic reticulum
  • cGAMP cyclic cGMP-AMP dinucleotide synthase
  • STING cyclic cGMP-AMP dinucleotide synthase
  • cGAMP stimulates the induction of interferon INF-I through STING, mediates the activation of TBK1 and IRF-3, and then initiates the transcription of type I interferon INF- ⁇ gene.
  • STING as a target immune activator/agonist, has anti-tumor, anti-AD and other diseases by stimulating the type I interferon INF- ⁇ gene.
  • STING agonist cyclic dinucleotide cGAMP is a secondary signaling molecule, its short metabolic cycle in the body and instability caused by easy degradation limit its efficacy and druggability in clinical trials.
  • AD Alzheimer's Disease
  • US FDA and China CFDA Drugs for silent disease are very rare, and most of them treat the symptoms but not the root cause, and have insignificant efficacy.
  • Most of the world's anti-Alzheimer's disease drugs are still in preclinical and clinical stages, and they mainly act on neural signaling pathways and A ⁇ amyloid plaques.
  • drugs there are many patients and the demand for drugs is great.
  • the prevalence of AD among those aged 65 and above reaches 4.8%. For every 5 years of age, the prevalence doubles. Among those aged 85 and above, the prevalence of AD reaches 28.9%.
  • AD Alzheimer's disease was first reported by German scholar Alosi Alzheimer in 1907.
  • the most typical pathological characteristics of AD are: the occurrence of a large number of amyloid plaques in the cerebral cortex and hippocampus - namely senile plaques (SP), neurofibrillary tangles (NFTs), reduced number of neurons and empty granules. Bubble degeneration.
  • SP senile plaques
  • NFTs neurofibrillary tangles
  • the pathogenesis of AD is very complex and may be the result of the interaction of multiple factors.
  • AD amyloid-beta peptide
  • APP amyloid precursor protein
  • brain homeostasis regulatory proteins and their related metal ion homeostasis are closely related to the occurrence and development of AD.
  • chronic inflammation of the brain is one of the distinctive features of AD.
  • a technological study from the University of Bonn in Germany has discovered that Alzheimer's disease is caused by inflammation of immune cells in the brain. Previously, humans have not been able to fully determine the cause and pathogenesis of Alzheimer's disease. This discovery is undoubtedly a major breakthrough.
  • Cyclic dinucleotide cGAMP has been reported as a natural immune agonist of STING It shows the efficacy of treating AD, improving the learning and memory ability of AD model mice, reducing amyloid plaques in the brain, reducing chronic inflammation in the brain, etc.
  • the immune agonist cGAMP is a secondary signaling molecule and is metabolized very quickly in the body, seriously affecting its efficacy and duration.
  • the object of the present invention is to overcome the shortcomings of the prior art and provide a cyclic dinucleotide metal compound, which is composed of a cyclic dinucleotide and metal ions (including lithium, magnesium, zinc, manganese , iron) metal compounds formed.
  • cyclic dinucleotide metal compounds overcome the shortcomings of rapid metabolism in the body, significantly extend their metabolism cycle in the body, and significantly increase their stability in the body. In mouse disease model tests, they show better performance than cyclic dinucleotides themselves. More significant anti-disease activity, including anti-Alzheimer's disease, ischemic brain injury, tumors and other diseases. Therefore, this type of innovative cyclic dinucleotide metal compounds has broad application prospects in the preparation of anti-disease drugs.
  • cyclic dinucleotide metal compounds the structural formula of the compound is [M (cyclic dinucleotide)] or [M' 2 (cyclic dinucleotide)] , wherein M is Mg 2+ , Zn 2+ , Mn 2+ or Fe 2+ , the M' is Li + , and the cyclic dinucleotide is 2'3'-cGAMP, c-di-AMP, Any one of c-di-GMP, c-di-IMP, c-GMP-IMP and derivatives thereof.
  • M in the compound is Mg 2+ .
  • the second object of the present invention is to provide a method for preparing a cyclic dinucleotide metal compound, which includes the following steps: binding the cyclic dinucleotide anion to an ion exchange column, and using a salt solution gradient of the target metal ion to elute the ion exchange column, collect the eluted cyclic dinucleotide metal compound solution, concentrate and desalt, and freeze-dry or cool to crystallize to precipitate the target product.
  • the present invention also provides the application of cyclic dinucleotide metal compounds in the preparation of drugs for preventing and treating neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis. disease, Huntington's disease.
  • the cyclic dinucleotide metal compound is lithium cyclic dinucleotide.
  • the present invention also provides the application of cyclic dinucleotide metal compounds in preparing drugs for preventing and treating ischemic cerebrovascular injury or craniocerebral injury.
  • the cyclic dinucleotide metal compound is magnesium cyclic dinucleotide.
  • the present invention also provides the application of cyclic dinucleotide metal compounds in the preparation of anti-tumor drugs and drugs for preventing and treating toxic and side effects caused by anti-tumor drugs.
  • the present invention also provides the application of cyclic dinucleotide metal compounds in the preparation of anti-coronavirus and viral inflammatory drugs and anti-viral vaccines.
  • the dosage form of the drug is tablets, capsules, enteric particles, granules, suspensions, emulsions, solutions, syrups or injections.
  • the cyclic dinucleotide metal compound of the present invention forms a stable metal compound structure through ionic bonds between two negatively charged phosphate groups in the cyclic dinucleotide and metal ions to extend its metabolic cycle in the body. , increasing its efficacy concentration when used as a drug, having the advantages of simple structure and broad application prospects in the preparation of anti-disease drugs.
  • the cyclic dinucleotide metal compound of the present invention is prepared through an ion exchange column.
  • the preparation method is simple and easy to operate.
  • the prepared cyclic dinucleotide metal compound has high purity and high purity. It has low mass content and is convenient for the preparation of related drugs.
  • the cyclic dinucleotide metal compound of the present invention not only has significant anti-disease activity in the preparation of drugs for preventing and treating neurodegenerative diseases, ischemic cerebrovascular injury or craniocerebral injury, anti-tumor drugs and anti-viral drugs, etc., Among them, lithium cyclic dinucleotide is particularly effective in preparing drugs for preventing and treating neurodegenerative diseases, and magnesium cyclic dinucleotide is used in preparing drugs for preventing and treating ischemic cerebrovascular injury or craniocerebral injury.
  • Figure 1 is the structural formula of the cyclic dinucleotide metal compound of the present invention.
  • Figure 2 is NMR of the products prepared in Examples 1, 2 and 5 of the present invention.
  • Figure 3 is the NMR of the products prepared in Examples 3 and 4 of the present invention.
  • Figure 4 is an MS chart of the cyclic dinucleotide compound of the present invention.
  • the present invention provides a cyclic dinucleotide metal compound.
  • the structural formula of the compound is [M (cyclic dinucleotide)] or [M' 2 (cyclic dinucleotide)], wherein M in the structural formula can be Mg 2+ , Zn 2+ , Mn 2+ or Fe 2+ and other positive divalent metal ions, especially M is the cyclic dinucleotide magnesium formed by Mg 2+ ; M' in the structural formula can be positive monovalent metal ions such as Li + Metal ion.
  • the cyclic dinucleotide is any one of 2'3'-cGAMP, c-di-AMP, c-di-GMP, c-di-IMP, c-GMP-IMP and derivatives thereof.
  • the The cyclic dinucleotides are all 2'3'-cGAMP (C 20 H 22 N 10 O 13 P 2 Na 2 ).
  • the dosage form of the drug can be tablets, capsules, enteric particles, granules, suspensions, emulsions, solutions, syrups or injections.
  • This example provides a method for preparing lithium cyclic dinucleotide: Dissolve cyclic dinucleotide (10 mmol) in 1L purified water, use AKTA Pure protein purification instrument, and pass the cyclic dinucleotide aqueous solution through an anion exchange column (SOURCE Q, GE Healthcare) to bind cyclic dinucleotides to SOURCE Q anion exchange columns. Wash the SOURCE Q column with 3 times the column volume of 20mM lithium chloride solution, and then gradient elute with lithium chloride aqueous solution (molar gradient concentration is 50mM ⁇ 300mM). The analyzed lithium chloride used in this example was purchased from Sigma.
  • lithium cyclic dinucleotide metal compound [Li 2 (cGAMP)] solution collect the eluted cyclic dinucleotide metal compound [Li 2 (cGAMP)] solution.
  • the lithium cyclic dinucleotide obtained by elution is concentrated with a nanofiltration membrane.
  • the nanofiltration membrane filters out the excess lithium chloride in the solution, adds ultrapure water and then concentrates. Repeat more than 5 times, and finally concentrate to 1/5 ( The total volume of the original eluent) was dried with a freeze dryer, and the yield of the obtained lithium cyclic dinucleotide was 78%.
  • the structure of lithium cyclic dinucleotide is shown in Figure 1.
  • the metal content analysis, elemental analysis, and NMR and MS characterization of lithium cyclic dinucleotide were performed. The analysis results are shown in Table 1.
  • the NMR data are shown in Figure 2.
  • the MS chart of lithium cyclic dinucleotide is shown in Figure 3.
  • the ESI-MS result shows that the target product peak is 673.0906, which is the ion peak of the cyclic dinucleotide anion. That is, the lithium cyclic dinucleotide prepared in this example is In the form of metal salts, they exist in solution as metal salt ions and cyclic dinucleotide ions respectively.
  • this embodiment prepares magnesium cyclic dinucleotide, and the eluent uses magnesium chloride solution. After the collected magnesium cyclic dinucleotide solution is concentrated, it is dried at 4°C. The cyclic dinucleotide magnesium compound was crystallized by cooling at low temperature, and was obtained after drying in a constant temperature oven. The yield of the obtained cyclic dinucleotide magnesium was 81%.
  • the structure of magnesium cyclic dinucleotide is shown in Figure 1. Metal content analysis, elemental analysis, and NMR and MS characterization were performed on magnesium cyclic dinucleotide. The analysis results are shown in Table 1. Its nuclear magnetic data is consistent with that of lithium cyclic dinucleotide. are basically consistent, and the MS pattern is also consistent with that of lithium cyclic dinucleotide.
  • this embodiment prepares manganese cyclic dinucleotides, manganese chloride is used as the eluent, and the yield of manganese cyclic dinucleotides is 78%.
  • the structure of cyclic dinucleotide manganese is shown in Figure 1.
  • Metal content analysis, elemental analysis, NMR and MS characterization were performed on cyclic dinucleotide manganese. The analysis results are shown in Table 1. Since the 3d orbital of manganese ions has a single electron and is paramagnetic, a certain amount of EDTA must be added to eliminate the interference of Mn 2+ paramagnetic ions during nuclear magnetic analysis.
  • this embodiment prepares cyclic dinucleotide iron, the eluent uses ferrous sulfate (containing sodium dithionite reducing agent), and the product of the obtained cyclic dinucleotide iron The rate is 75%.
  • the structure of cyclic dinucleotide iron is shown in Figure 1.
  • the cyclic dinucleotide iron was subjected to metal content analysis, elemental analysis, and NMR and MS characterization. Since the 3d orbital of the iron ion has a single electron and is paramagnetic, it is in progress. During NMR analysis, a certain amount of EDTA must be added to eliminate the interference of Fe 2+ paramagnetic ions.
  • the analysis results are shown in Table 1.
  • the NMR data is basically consistent with that of manganese cyclic dinucleotide, and its MS pattern is also consistent with that of lithium cyclic dinucleotide. consistent.
  • Example 1 The main difference between this example and Example 1 is that this example prepares zinc cyclic dinucleotide, and the eluent uses zinc chloride solution (the pH value is adjusted to weakly acidic ⁇ 6). The obtained cyclic dinucleotide The zinc yield was 72%.
  • the structure of cyclic dinucleotide zinc is shown in Figure 1. Metal content analysis, elemental analysis, and NMR and MS characterization were performed on cyclic dinucleotide magnesium. The analysis results are shown in Table 1. Its nuclear magnetic data is consistent with that of cyclic dinucleotide lithium. are basically consistent, and the MS pattern is also consistent with that of lithium cyclic dinucleotide.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 1 to 5 in preparing drugs for preventing and treating neurodegenerative diseases, and mainly verifies the effects of the cyclic dinucleotide metal compounds on the cognitive ability of AD mice.
  • AD mice were randomly divided into 7 groups, with 10 rats in each group.
  • the 7 groups were as follows: A: AD model group, as the negative control group (administration: physiological saline); B: cGAMP group, as the positive control group (administration: Cyclic dinucleotide); C: [Li 2 (cGAMP)] group (administration: lithium cyclic dinucleotide prepared in Example 1); D: [Mg (cGAMP)] group (administration: Example 2 Cyclic dinucleotide magnesium prepared); E: [Zn(cGAMP)] group (administration: cyclic dinucleotide zinc prepared in Example 5); F: [Mn(cGAMP)] group (administration: implementation Cyclic dinucleotide manganese prepared in Example 3); G: [Fe(cGAMP)] group (administration: cyclic dinucleotide iron prepared
  • the preparation method of the drugs given to the CG group is: use physiological saline to
  • the powder prepared in the example should be prepared into a solution of required concentration.
  • the dosage of each group is 10 mg/kg.
  • the dosage method is intraperitoneal injection.
  • the number of dosages is: once every 2 days for 60 consecutive days. .
  • the sub-pools are There are four quadrants, and a platform is placed in the center of the third quadrant (the distance between the platform and the center of the pool wall is equal); it is submerged 1cm underwater to make the platform invisible. Rich reference cues (triangles, squares, circles, diamonds of different colors placed in each quadrant) were posted around the pool and remained unchanged for mice to use to locate the platform.
  • Positioning navigation test The test lasted for 6 days, with 4 training sessions scheduled at a fixed time every day. At the beginning of training, place the platform in the first quadrant, and put the mouse into the water facing the pool wall from any of the four starting points on the pool wall.
  • the free video recording system records the time it takes for the mice to find the platform and the swimming path.
  • the mice are put into the water from four different starting points (different quadrants) in four training sessions. After the mouse finds the platform or cannot find the platform within 90 seconds (the latency period is recorded as 90 seconds), the experimenter will guide it to the platform and rest on the platform for 10 seconds before conducting the next test.
  • the positive control group B using cyclic dinucleotides and the C-G group using cyclic dinucleotide metal compounds can significantly improve the cognitive ability of Alzheimer's mice after 60 days of administration.
  • Groups C-G (cyclic dinucleotide metal compound administration group) improved cognitive ability of AD mice significantly better than group B (cyclic dinucleotide positive control group).
  • group C (cyclodinucleotide positive control group) The improvement effect of lithium nucleotide administration group) is significantly better than that of other cyclic dinucleotide metal compounds.
  • the efficacy of applying lithium cyclic dinucleotide to prepare drugs for preventing and treating neurodegenerative diseases is particularly outstanding, showing more Excellent drug efficacy improvement.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 1 to 5 in preparing drugs for preventing and treating neurodegenerative diseases, and mainly verifies the effects of the cyclic dinucleotide metal compounds on brain amyloid plaques in AD mice.
  • Example 6 The main difference between this embodiment and Example 6 is that after 60 days of administration to seven groups of AD model mice, the reduction of amyloid plaques in the brains of AD mice was detected.
  • the experiment conducted in this example was a thioflavin S staining experiment.
  • the experimental process was as follows: 60 days after administration, the mouse brain group was taken. Tissue, fixed, paraffin embedded, sectioned, xylene dewaxed, ethanol gradient dehydration, washed three times with TBS, 0.3% thioflavin S (dissolved in 50% ethanol) was dropped on the tissue, incubated at room temperature for 10 min, washed three times with 50% ethanol. TBS was cleared, dried in the shade, mounted, and confocal laser microscopy (Leica, Germany) was used to detect changes in the amount of amyloid plaque deposition in the brains of AD mice. The experimental results are shown in Table 3.
  • the positive control group B using cyclic dinucleotides and the C-G group using cyclic dinucleotide metal compounds can significantly reduce amyloid in the brain tissue of Alzheimer's mice after 60 days of administration.
  • the improvement effect of lithium cyclic dinucleotide (group C) is particularly outstanding.
  • the application of lithium cyclic dinucleotide to prepare drugs for the prevention and treatment of neurodegenerative diseases The drug efficacy shows a better improvement in drug efficacy.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 1 to 5 in preparing drugs for preventing and treating ischemic cerebrovascular injury, and mainly verifies the effects of cyclic dinucleotide metal compounds on mice with ischemic cerebrovascular injury. .
  • mice Healthy male ICR mice, weighing 18-20 grams, were purchased from Shanghai Slack Experimental Animal Co., Ltd., quality certificate number (SCXK (Shanghai) 2007-0005), and were raised in a detergent animal room.
  • SCXK quality certificate number
  • Mouse model Mouse suture method was used to create a local cerebral ischemia model to verify the therapeutic effect of cyclic dinucleotide metal compounds on ischemic brain disease in experimental animals.
  • mice were anesthetized with 10% chloral hydrate intraperitoneally, and a midline incision was made on the neck.
  • the proximal segment of the right common carotid artery, carotid artery and its branch vessels were isolated and ligated.
  • the right internal carotid artery was isolated, the pterygopalatine artery was isolated along the internal carotid artery downward, and the branch was ligated at the root.
  • the suture enters the internal carotid artery and enters the cranium to the anterior cerebral artery to block all blood flow in the middle cerebral artery. source.
  • the arterial clamp was removed, sutures were inserted, the skin was sutured, and the animals were returned to the cage for rearing after surgery.
  • intraperitoneal injection was administered, the nylon suture was pulled out for reperfusion, and 8 hours of reperfusion was followed before administration.
  • Behavioral scoring was performed after surgery. The scoring was performed using a single-blind method. The scoring was based on the Zea Longa 5-point scoring standard.
  • the scoring method is as follows: 0 points, the mouse is normal and has no symptoms of nerve damage; 1 point, the contralateral forepaw cannot be fully extended; 2 points, turning in circles to the outside; 3 points, falling over relative to the test; 4 points, unable to walk spontaneously, loss of consciousness.
  • mice were divided into 7 groups, with 10 mice in each group.
  • the 7 groups were: A: AD model group, as the negative control group (administration: physiological saline); B: cGAMP group, as the positive control group (administration: physiological saline) : cyclic dinucleotide); C: [Li 2 (cGAMP)] group (administration: cyclic dinucleotide lithium prepared in Example 1); D: [Mg (cGAMP)] group (administration: Example Cyclic dinucleotide magnesium prepared in Example 2); E: [Zn(cGAMP)] group (administration: cyclic dinucleotide zinc prepared in Example 5); F: [Mn(cGAMP)] group (administration: Cyclic dinucleotide manganese prepared in Example 3; G: [Fe(cGAMP)] group (administration: cyclic dinucleotide iron prepared in Example 4).
  • the preparation method of the drugs given to the CG group is: use physiological saline to prepare the powder prepared in the corresponding example into a solution with the required concentration.
  • the dosage of each group is 10 mg/kg, and the administration method is intraperitoneal injection.
  • One dose was administered after 2 hours of ischemia, the nylon thread was pulled out for reperfusion, and another dose was administered after 8 hours of reperfusion. After 24 hours, administer once a day for 7 days.
  • the experimental results of each group are shown in Table 4.
  • intraperitoneal injection administration of groups B-G can improve the behavioral scores of mice with focal cerebral ischemia.
  • the cyclic dinucleotide metal compounds The drug administration group (Group C-G) has a more obvious medicinal effect on ischemic cerebrovascular injury.
  • the improvement effect of magnesium cyclic dinucleotide (Group D) is particularly outstanding, and the medicine prepared by it to prevent and treat ischemic cerebrovascular injury It clearly has significantly improved medicinal efficacy.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 1 to 5 in preparing drugs for preventing and treating ischemia-reperfusion injury, and mainly verifies the effects of the cyclic dinucleotide metal compounds on mice with ischemia-reperfusion injury.
  • Rat cerebral ischemia model establishment and experimental methods 280-300g male SD rats were selected for the experiment. They were fasted and not allowed to drink water for 12 hours before the operation. They were weighed, numbered and randomly divided into groups. Anesthetize the rat by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL/100 g. After the rat has no pain reflex, prepare the skin on the neck and fix it supine on the rat board. After sterilizing the neck skin with sterilized cotton balls, place it along the middle of the neck. Cut about 1cm of skin to expose the subcutaneous tissue. Use curved forceps to bluntly remove the subcutaneous fat and fascia to expose the neck muscles.
  • Drug preparation and experimental grouping The positive control drug is edaravone, the specification of edaravone injection is 30mg/20mL, and the single dose of rat is 0.4mL/100g, that is, the single dose of 6mg/kg .
  • the cyclic dinucleotide metal complexes prepared in Examples 1 to 5 were formulated into injection drugs with a concentration of 0.75 mg/mL using physiological saline, and each rat was given a dose of 0.4 mL/100 g of drug through the tail vein, that is, a single dose. is 5mg/kg.
  • the rats were divided into 8 groups, with 10 rats in each group.
  • the 8 groups were as follows: model group: normal saline, injected into the tail vein at 4 mg/kg; positive control group: edaravone, injected into the tail vein at 6 mg/kg. ; Group I: cyclic dinucleotide, 5 mg/kg tail vein injection; Group II: [Li 2 (cGAMP)], 5 mg/kg tail vein injection; Group III: [Mg (cGAMP)], 5 mg/kg kg tail vein injection; Group IV: [Zn(cGAMP)], 5 mg/kg tail vein injection; Group V: [Mn(cGAMP)], 5 mg/kg tail vein injection; Group VI: [Fe(cGAMP)] , injected into the tail vein at 5 mg/kg. Each rat in the drug group was injected into the tail vein 30 minutes after ischemia. The statistical results of TTC staining of rat brain tissue in each group are shown in Table 5.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 1 to 5 in the preparation of anti-tumor drugs, and mainly verifies the inhibitory effect of the cyclic dinucleotide metal compounds on the growth of transplanted tumors in mice.
  • mice BALB/C ordinary mice, C57BL/6 ordinary mice, male, weighing 20-22 grams, 7-8 weeks old, purchased from Shanghai Slack Experimental Animal Co., Ltd.
  • Raising conditions All mice had free access to food and water, and were raised at room temperature (23 ⁇ 2)°C. Feed and water are all processed by high-pressure sterilization, and all experimental feeding processes are SPF grade.
  • Tumor cell lines mouse colorectal cancer cell line CT26, mouse breast cancer cell line 4T1, All were purchased from the Cell Bank of the Chinese Academy of Sciences.
  • mice cell culture, passaging, collecting cells in the logarithmic phase, making a cell suspension with a concentration of (1.0 ⁇ 10 7 ) per ml, and injecting 0.2 ml of cell suspension into the armpit of the right forelimb of the mouse (The number of cells is 2.0 ⁇ 10 6 /bird), and the tumor was successfully transplanted in about 7 days.
  • the mice with successful tumor transplantation were randomly divided into 7 groups, with 10 mice in each group.
  • the 7 groups were: A: negative control group (injection of normal saline), B: positive control cGAMP group (10 mg/kg), C: [ Li 2 (cGAMP)] group (10 mg/kg), D: [Mg (cGAMP)] group (10 mg/kg), E: [Zn (cGAMP)] group (10 mg/kg), F: [Mn (cGAMP) ] group (10 mg/kg), G: [Fe(cGAMP)] group (10 mg/kg).
  • the method of administration is intraperitoneal injection, the administration volume is 200 ⁇ l/animal, once every 2 days, for 20 consecutive days. After 20 days, the mice were sacrificed and the tumor weight was measured.
  • the tumor inhibition rate [1-average tumor weight of the experimental group/average tumor weight of the A negative control group] ⁇ 100%.
  • the mouse colorectal cancer cell line CT26 was cultured and transplanted into BAlB/C ordinary mice.
  • the mouse breast cancer cell line 4T1 was transplanted into BAlB/C ordinary mice to observe the anti-tumor effects of different drugs.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 3 and 5 in the preparation of drugs for preventing and treating toxic and side effects caused by anti-cancer drugs. It mainly verifies the toxic and side effects of cyclic dinucleotide metal compounds as anti-cancer drugs. reducing effect.
  • mice BALB/C ordinary mice, male, weighing 20-22 grams, 7-8 weeks old, SPF grade, purchased from Southern Model Biotechnology Co., Ltd.
  • Raising conditions All mice had free access to food and water, and were raised at room temperature (23 ⁇ 2)°C. Feed and water are all processed by high-pressure sterilization, and all experimental feeding processes are SPF grade.
  • the tumor cell line was the mouse colorectal cancer cell line CT26, which was purchased from the Cell Bank of the Chinese Academy of Sciences.
  • mice cell culture, passaging, collecting cells in the logarithmic phase, making a cell suspension with a concentration of (1.0 ⁇ 10 7 ) per ml, and injecting 0.2 ml of cell suspension into the armpit of the right forelimb of the mouse (The number of cells is 2.0 ⁇ 10 6 /bird), and the tumor was successfully transplanted in about 7 days.
  • the mice were randomly divided into 5 groups, with 10 mice in each group.
  • the 5 groups were: A: negative control group (injection of normal saline), B: positive control oxaliplatin group (5mg/kg), C: oxaliplatin Platinum (5mg/kg) + cGAMP (10mg/kg), D: oxaliplatin (5mg/kg) + [Mn (cGAMP)] (10mg/kg), E: oxaliplatin (5mg/kg) + [Zn(cGAMP)](10mg/kg).
  • the method of administration is intraperitoneal injection, the administration volume is 200 ⁇ l/animal, once every 2 days, for 20 consecutive days. After 20 days, weigh the mice, take blood, and then sacrifice the mice and weigh the tumors.
  • the tumor inhibition rate [1-average tumor weight of the experimental group/average tumor weight of the negative control group] ⁇ 100%.
  • mice Effects of cyclic dinucleotide metal compounds on the body weight and survival rate of mice: The weight loss percentage, survival rate and average tumor inhibition rate of mice were calculated after 20 days of administration. The statistical results are shown in Table 8.
  • cyclic dinucleotides and their metal compounds [Mn(cGAMP)] and [Zn(cGAMP)] significantly reduce the toxic and side effects of platinum metal complexes.
  • the weight of the positive control oxaliplatin group decreased by 40%; the combination of oxaliplatin and cyclic dinucleotide metal compounds was much less toxic than oxaliplatin alone, and the survival rate and tumor inhibition rate were significantly improved.
  • Manganese dinucleotide and zinc cyclic dinucleotide have a significant effect on improving the toxic and side effects of anticancer drugs, achieving the effect of increasing efficacy and reducing toxicity.
  • Cyclic dinucleotide metal compounds reduce the blood toxicity of anti-tumor drugs: Peripheral blood was collected from each group of mice in the experiment, and the results of routine blood tests were compared. The test results are shown in Table 9.
  • Cyclic dinucleotide metal compounds reduce the toxicity of anti-tumor drugs to the liver and kidneys of mice: The contents of alanine aminotransferase (ALT) and creatinine (Creatinine) in the serum of mice in each group were statistically calculated. The results are shown in Table 10.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 1 to 5 as immune adjuvants in the preparation of antiviral vaccines, mainly to verify the immune adjuvant function of the cyclic dinucleotide metal compounds.
  • mice BALB/C ordinary mice, male, weighing 20-22 grams, 7-8 weeks old, SPF grade, purchased from Shanghai Slack Experimental Animal Co., Ltd.
  • Raising conditions All mice had free access to food and water, and were raised at room temperature (23 ⁇ 2)°C. Feed and water are all processed by high-pressure sterilization, and all experimental feeding processes are SPF grade.
  • mice were randomly divided into 8 groups, with 10 mice in each group.
  • the 8 groups were: A: Negative control group; B: OVA+aluminum adjuvant; C: OVA+cyclic dinucleotide; D: OVA+[Li 2 (cGAMP)]; E: OVA+[Mg(cGAMP)]; F: OVA+[Zn(cGAMP)] ; G: OVA+[Mn(cGAMP)]; H: OVA+[Fe(cGAMP)].
  • mice in the BH group were subcutaneously injected with 10 ⁇ g of OVA and 100 ⁇ g of aluminum adjuvant or cyclic dinucleotide or its metal compound, and the negative control group was injected with physiological saline.
  • the mice were immunized once on days 1, 7, and 14, and lung lavage fluid and blood samples were obtained on day 21.
  • the ELISA method was used to determine the potency of cyclic dinucleotide metal compounds used as adjuvants to induce the production of antibodies.
  • the experimental results are shown in Table 11.
  • cyclic dinucleotides and their metal compounds can significantly induce immune antibodies, and the effect is significantly better than aluminum adjuvants.
  • [Mn(cGAMP)] and [Zn(cGAMP)] have outstanding immune adjuvant effects, which are more significantly improved than cyclic dinucleotides and other metal complexes.
  • This example provides the application of the cyclic dinucleotide metal compounds prepared in Examples 1 to 5 in the preparation of antiviral inflammatory drugs, mainly to verify that the cyclic dinucleotide metal compounds induce Activates adaptive immunity.
  • mice BALB/C ordinary mice, C57BL/6 ordinary mice, male, weighing 20-22 grams, 7-8 weeks old, purchased from Shanghai Slack Experimental Animal Co., Ltd.
  • Raising conditions All mice had free access to food and water, and were raised at room temperature (23 ⁇ 2)°C. Feed and water are all processed by high-pressure sterilization, and all experimental feeding processes are SPF grade.
  • mice were randomly divided into 8 groups, with 10 mice in each group.
  • the 8 groups were: A: negative control group (isotype control antibody group), B: blank group, C: cyclic dinucleotide group, D: [Li 2 (cGAMP)] group, E: [Mg(cGAMP)] group, F: [Zn(cGAMP)] group, G: [Mn(cGAMP)] group (10 mg/kg), H: [Fe(cGAMP)] Group.
  • the isotype control flow cytometry antibody was purchased from eBiosciences, the antibody magnetic strain was purchased from Militeny Biotech, and the flow cytometer was purchased from BD.
  • the mouse spleen and lung tissues were taken and ground and pounded respectively. Crush the cells, filter them through 40 micron holes, and centrifuge at 1000 rpm for 10 minutes to separate unlysed immune cells. Use antibody magnetic strains to separate DC (CD40 ⁇ CD80 ⁇ CD86 ⁇ MHCII) and T (CD8+) cells, and add the corresponding FAC antibodies. (diluted with FACS buffer), and the isotype control antibody was used as a negative control.
  • cyclic dinucleotide metal compounds can significantly activate dendritic cells DC and T cells.
  • [Mn(cGAMP)] and [Zn(cGAMP)] have outstanding pharmacodynamic effects in inducing acquired immunity. , has a more significant improvement than cyclic dinucleotides and other metal complexes.
  • mice purchased from Shanghai Slack Experimental Animal Co., Ltd., half male and half female, weighing 20-22g. The animals were fed with pellet feed and had free access to food and water.
  • mice were intraperitoneally injected with 1g/kg cyclic dinucleotide metal compound drug (prepared with normal saline for injection) according to body weight, and the toxic reactions and death of the mice within 14 days after administration were observed. The results showed that after intraperitoneal injection of the drug, the mice's activities were normal. No mice died within 14 days after administration. On the 15th day, all mice were sacrificed, dissected, and each organ was visually inspected. No obvious lesions were found.
  • mice SD rats, weighing 200-220g, were purchased from Shanghai Slack Experimental Animal Co., Ltd.
  • Raising conditions All rats had free access to food and water, and were raised at room temperature (25 ⁇ 2)°C. Feed and water are all processed by high-pressure sterilization, and all experimental feeding processes are SPF grade.
  • Rat ELISA kit was used to determine the concentrations of cGAMP and IFN- ⁇ in serum. Based on the activation of natural immune pathways by cGAMP or cyclic dinucleotide metal compounds, the production of IFN- ⁇ is induced.

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Abstract

La présente invention relève du domaine technique des médicaments biologiques. L'invention concerne un composé métallique dinucléotidique cyclique, son procédé de préparation et son utilisation. La formule développée du composé est [M(dinucléotide cyclique)] ou [M'2(dinucléotide cyclique)], M étant Mg2+, Zn2+, Mn2+ ou Fe2+, M' étant Li+, et le dinucléotide cyclique étant l'un quelconque parmi 2'3'-cGAMP, c-di-AMP, c-di-GMP, c-di-IMP, c-GMP-IMP et leurs dérivés. Le composé métallique dinucléotidique cyclique a un cycle métabolique in vivo plus long et une meilleure stabilité in vivo ; par comparaison avec le dinucléotide cyclique en soi, le composé métallique dinucléotidique cyclique a une activité contre des maladies plus remarquable ; le composé métallique dinucléotidique cyclique présente les avantages d'une structure simple et d'une large perspective d'application dans la préparation de médicaments contre des maladies.
PCT/CN2023/109735 2022-09-14 2023-07-28 Composé métallique dinucléotidique cyclique, son procédé de préparation et son utilisation WO2024055758A1 (fr)

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